Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (SHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.
GFAP; Mutation; Alexander disease; Intermediate filament; Stress; Small heat shock protein
Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/μm2; equator: 8.2 ± 1.9 particles/μm2; posterior pole: 4 ± 0.9 particles/μm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/μm2; equator: 5 ± 2 particles/μm2; posterior pole: 1.9 ± 0.7 particles/μm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.
lens capsule; FGF-2; perlecan; ERK1/2
IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD225 in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp225 produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.
Alexander disease; caspase; GFAP; intermediate filament; AD, Alzheimer’s disease; AxD, Alexander disease; C-GFAP, C-terminal GFAP; CNS, central nervous system; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; IF, intermediate filament; N-GFAP, N-terminal GFAP OA, okadaic acid; TBS, Tris-buffered saline; TBST, TBS containing 0.1% (v/v) Tween 20
In bony fishes, Bfsp2 orthologues are predicted to possess a C-terminal tail domain, which is absent from avian, amphibian and mammalian Bfsp2 sequences. These sequences, are however, not conserved between fish species and therefore questions whether they have a functional role. For other intermediate filament proteins, the C-terminal tail domain is important for both filament assembly and regulating interactions between filaments. We confirm that zebrafish has a single Bfsp2 gene by radiation mapping. Two transcripts (bfsp2α and bfsp2β) are produced by alternative splicing of the last exon. Using a polyclonal antibody specific to a tridecameric peptide in the C-terminal tail domain common to both zebrafish Bfsp2 splice variants, we have confirmed its expression in zebrafish lens fibre cells. We have also determined the in vitro assembly properties of zebrafish Bfsp2α and conclude that the C-terminal sequences are required to regulate not only the diameter and uniformity of the in vitro assembly filaments, but also their filament–filament associations in vitro. Therefore we conclude zebrafish Bfsp2α is a functional orthologue conforming more closely to the conventional domain structure of intermediate filament proteins. Data mining of the genome databases suggest that the loss of this tail domain could occur in several stages leading eventually to completely tailless orthologues, such as human BFSP2.
lens; cytoskeleton; beaded filaments; BFSP1; intermediate filament; evolution
The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na+-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na+/K+-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na+-driven influx of solutes occurs via paracellular and membrane transport and the Na+/K+-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.
vertebrate eye lens; solute flux; ultrastructure; gap junctions; MRI; dye loading
The β3- and β8-strands and C-terminal residues 155–165 of αB-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised αB-crystallin protein constructs with substituted β3- and β8-strands and with the C-terminal residues 155–165 deleted to demonstrate the importance of these sequences to the interaction of αB-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of αB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured αB-crystallin cosedimentation with desmin filaments. Swapping the β8-strand of αB-crystallin or deleting residues 155–165 increased the cosedimentation of αB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the β3-strand with the equivalent αA-crystallin sequences improved the ability of αB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (β3-strand, β8-strand and C-terminal residues 155–165) contribute to the interaction of αB-crystallin with desmin filaments. The data also suggest that the cosedimentation of αB-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and αB-crystallin and typify desmin related myopathies, but also to the interaction of αB-crystallin with other filamentous protein polymers.
MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and αB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2−/− compared to MK2+/+ hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and αB crsytallin phosphorylation were reduced to baseline in MK2−/− hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2+/+ hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2+/+ hearts was lost in MK2−/− hearts, it was basally elevated in nuclei of MK2−/− hearts and was similar to that seen during ischemia in MK2+/+ hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2+/+ and MK2−/− hearts, which were decreased by the p38 inhibitor SB203580 (1 μM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2−/− hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury independently of MK2.
Ischemia; MAPKAPK-2; p38 MAPK; HSP27; αB-crystallin
Intermediate filaments (IFs) are a key component of the cytoskeleton in virtually all vertebrate cells, including those of the lens of the eye. IFs help integrate individual cells into their respective tissues. This Review focuses on the lens-specific IF proteins beaded filament structural proteins 1 and 2 (BFSP1 and BFSP2) and their role in lens physiology and disease. Evidence generated in studies in both mice and humans suggests a critical role for these proteins and their filamentous polymers in establishing the optical properties of the eye lens and in maintaining its transparency. For instance, mutations in both BFSP1 and BFSP2 cause cataract in humans. We also explore the potential role of BFSP1 and BFSP2 in aging processes in the lens.
The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical–basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial–mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.
Lens; aPKC; Polarity; Cell junction; Proliferation
Microtubules are self-assembling polymers whose dynamics are essential for the
normal function of cellular processes including chromosome separation and
cytokinesis. Therefore understanding what factors effect microtubule growth is
fundamental to our understanding of the control of microtubule based processes.
An important factor that determines the status of a microtubule, whether it is
growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we
derive a Monte Carlo model of the assembly and disassembly of microtubules. We
use thermodynamic laws to reduce the number of parameters of our model and, in
particular, we take into account the contribution of water to the entropy of the
system. We fit all parameters of the model from published experimental data
using the GTP tubulin dimer attachment rate and the lateral and longitudinal
binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate
and incorporate the GTP hydrolysis rate. We have applied our model and can mimic
published experimental data, which formerly suggested a single layer GTP tubulin
dimer microtubule cap, to show that these data demonstrate that the GTP cap can
fluctuate and can be several microns long.
Here we review how GFAP mutations cause Alexander disease. The current data suggest that a combination of events cause the disease. These include: i) the accumulation of GFAP and the formation of characteristic aggregates, called Rosenthal fibres, ii) the sequestration of the protein chaperones αB-crystallin and HSP27 into Rosenthal fibres, and iii) the activation of both Jnk and the stress response. These then set in motion events that lead to Alexander disease. We discuss parallels with other intermediate filament diseases and assess potential therapies as part of this review as well as emerging trends in disease diagnosis and other aspects concerning GFAP.
Alexander disease; GFAP; chaperone; alphaB-crystallin; Jnk; MLK
The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-α, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-δ. GFAP-δ has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-α, assembly was restored providing GFAP-δ levels were kept low (∼10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-δ is naturally part of the endogenous intermediate filaments, although levels were low (∼10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease–causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-δ and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of αB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-δ splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein– and filament–filament associations as well as Jnk phosphorylation.
The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.
The R120G mutation in αB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of αB-crystallin for desmin filaments. The apparent dissociation constant of R120G αB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G αB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type αB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G αB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in αB-crystallin.
The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.
γ-Crystallin genes are specifically
expressed in the eye lens. Their promoters constitute excellent models
to analyse tissue-specific gene expression. We investigated murine Cryge/f promoters of different
length in lens epithelial cell lines. The most active fragment extends
from position –219 to +37. Computer analysis predicts
homeodomain and paired-domain binding sites for all rodent Crygd/e/f core promoters. As
examples, we analysed the effects of Prox1 and Six3, which are considered important
transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a
weak stimulation of Cryge/f promoter
activity was found for PROX1. In contrast, PROX1 stimulated the Crygf promoter 10-fold in CD5A cells without
endogenous PROX1. In both cell lines Six3 repressed
the Crygf promoter to 10% of its basal
activity. Our cell transfection experiments indicated that Cryg expression
increases as Six3 expression decreases. Prox1 and
Six3 act antagonistically on regulation of the Crygd/e/f promoters. Functional
assays using randomly mutated γF-crystallin promoter
fragments define a Six3-responsive element between –101
and –123 and a Prox1-responsive element between –151
and –174. Since Prox1 and Six3 are present at the beginning
of lens development, expression of Crygd/e/f is predicted to remain low
at this time. It increases as Six3 expression decreases during ongoing